Developing a robust in vivo hairy root system for assessing transgene expression and genome editing efficiency in papaya

Papaya is one of the most important fruits in tropical and subtropical countries. However, genetic improvement has had limited success to date due time-consuming complex transformation regeneration technologies, as well a lack reproducible efficient transient gene expression assays. Here, we report development highly Rhizobium rhizogenes-based vivo hairy root system for evaluating transgene activity including CRISPR/Cas editing reagents Vietnamese papaya cultivar Linhan. To optimize parameters, introduced R. rhizogenes strain K599 into hypocotyls at 1-, 5- 10-mm below cotyledon nodes by needle using 5-, 7- 10-day old seedlings then monitored frequency formation 18 days post infection. We found that age distance infection site from node were inversely correlated with efficacy induction, being 5-day-old plants 1-mm best parameters. The etablished protocol was employed investigate gus reporter gene. Of tested roots, 47.22% positive GUS staining, which indicates high level transfer stability. Finally, dual guide RNA CRISPR/Cas9 cassette targeting eukaryotic translation initiation factor isoform 4E (eIF(iso)4E) screened events heteroduplex analysis Sanger sequencing. Our revealed 50% induced roots contained expected mutations eIF(iso)4E gene, makes our ideal testing prior making stable transgenic lines. developed an procedure induction may be used validate accelerate CRISPR/Cas-based genome studies papaya.